The invention generally relates to the field of implantable diagnostic devices (i.e. devices deployed within the body of an animal) for monitoring one or more target substances, analytes, or metabolites in the animal. More particularly, the invention provides implantable biosensor devices for monitoring and regulating the level of analytes in the tissues and circulatory system of a human. In illustrative embodiments, the apparatus comprises a biosensor that is utilized to monitor the level of blood glucose in a diabetic or hypoglycemic patient. The disclosed sensors may also be used to control or regulate the delivery of a drug or other pharmaceutical agent from an external or an implantable drug delivery system. For example, the device may form part of an artificial pancreas to regulate insulin dosage in response to the level of glucose detected in situ.
1.2.1 Biosensors
Biosensors are hybrid devices combining a biological component with an analytical measuring element. The biological component reacts and/or interacts with the analyte(s) of interest to produce a response measurable by an electronic, optical, or mechanical transducer. The most common configurations presently available utilize immobilized macromolecules such as enzymes or antibodies to form the biological component. Examples of analytes and immobilized macromolecules include: glucose and immobilized glucose oxidase (e.g., Wilkins et al., 1995); nitrate and immobilized nitrate reductase (Wu et al., 1997); hydrogen peroxide and 2,3-dichlorophenoxyacetic acid and immobilized horseradish peroxidase (Rubtsova et al., 1998); and aspartate and immobilized L-aspartase (Campanella et al., 1995).
1.2.2 Whole-cell Biosensors
A further refinement for biosensors has been developed in recent years that utilizes intact living cells, such as a microorganism, or an eukaryotic cell or cell culture as an alternative to immobilized enzymes. Microbial cells are especially well suited for biosensor technologies; they are physically robust, capable of existing under extremely harsh and widely fluctuating environmental conditions, they possess an extensive repertoire of responses to their environment, and they can be genetically engineered to generate reporter systems that are highly sensitive to these environmental responses. Polynucleotide sequences that comprise specific promoter sequences are operably linked to a gene or a plurality of genes that encode the desired reporter enzyme(s) and then introduced into and maintained within the living cell. When the target analyte is present, the reporter genes are expressed, generating the enzyme(s) responsible for the production of the measured signal. Commonly used reporter systems have utilized either the xcex2-galactosidase (lacZ) or catechol-2,3-dioxygenase (xylE) enzymes (Kricka, 1993).
Unfortunately, a limitation of these systems has been that following exposure to the target substance(s), the cells must be destructively lysed and the enzyme(s) isolated. This lysis is then followed by the addition of one or more secondary metabolites to yield a colorimetric signal that is proportional to the concentration of enzyme(s) in solution, providing a means to quantify the concentration of the original target substance.
A more recent improvement in such sensors utilizes green fluorescent protein as a reporter system, with the significant advantage that cells do not require destructive assay techniques to produce colorimetric signals. Because a substrate must be added to the green fluorescent protein constructs to first initiate the light response, however, these systems are quite complicated and offer little advantage for detection of analytes in situ (Prasher, 1995).
1.23 In Vivo Sensors
The development of an integrated in vivo implantable glucose monitor was first reported by Wilkins and Atanasov (1995). This system utilizes glucose oxidase immobilized within a micro-bioreactor. This enzyme catalyzes the oxidation of xcex2-D-glucose by molecular oxygen to yield gluconolactone and hydrogen peroxide, with the concentration of glucose being proportional to the consumption of O2 or the production of H2Ohd 2. Unfortunately, the presence of a glucose oxidase inhibitor molecule in the human bloodstream tended to offset proportionality constants, and made the device unsatisfactorily inaccurate for precise glucose monitoring and control (Gough et al., 1997). Also limiting was the device""s relatively large size (≈5xc3x977 cm), which negated its usefulness as an implantable device.
Although several smaller needle-type and microdialysis glucose sensors have since been developed to circumvent size limitations (Gough et al., 1997, Selam, 1997), their reliance on a glucose oxidase enzyme-based system limits their overall effectiveness and reliability.
Several nonspecific electrochemical sensors have also been investigated as potential in vivo glucose sensors (e.g., Yao et al., 1994; Larger et al., 1994), but problems including limited sensitivity, instability, and limited long-term reliability have prevented their wide-spread utilization (Patzer et al., 1995). According to Atanasov et al. (1997), continuously functioning implantable glucose biosensors with long-term stability have yet to be achieved.
Despite a significant miniaturization of biosensors during the past decade, they are still relatively large and obtrusive to serve as ideal implantable devices. Current methodologies using mammalian bioluminescent reporter cells require cell lysis and addition of an exogenous substrate to generate a measurable response. Consequently, these cells cannot serve as continuous on-line monitoring devices.
Therefore, there remains a need for the development of a small implantable monolithic (i.e. containing both biological and electrical components constructed on a single substrate layer) bioelectronic monitor that is durable, inexpensive, wireless, and that can communicate remotely to a drug delivery system to provide the controlled delivery of a therapeutic agent such as insulin.
The present invention overcomes these and other inherent limitations in the prior art by providing implantable apparatus and methods for detecting and quantitating particular analytes in the body of an animal. In particular, the invention provides devices for the in vivo detection and quantitation of metabolites, drugs, hormones, toxins, or microorganisms such as viruses in a human or animal. In illustrative embodiments, the invention provides a BBIC device useful for the detection of glucose in a human. Such devices provide for the first time an accurate on-line detector for glucose monitoring, and offer the ability to control the administration of pharmaceutical agents via an external or implantable drug delivery system. Also disclosed are BBIC devices for detecting the concentration of signature molecules (i.e. proteins released from cancer cells, etc.), clotting factors, enzymes and the like, and other analytes present in the bloodstream or interstitial fluid. In the area of oncology, the biosensor devices find utility in both initial and remission monitoring, on-line measurement of the effectiveness of chemotherapy, and stimulation/activity of the immune system. Likewise, the biosensor devices are useful in other areas of medicine, including on-line monitoring for enzymes associated with the occurrence of blood clots (strokes, heart attacks, etc.), detection and quantitation of clotting factors (maintain level), hormone replacement, continuous drug monitoring (testing for controlled substances in prisoners, military personnel, etc.), monitoring of soldiers exposure to sub-lethal exposure to nerve agents and other debilitating agents, monitor levels of compounds affecting mental illness, and the like.
In one embodiment there is provided an implantable monolithic bioelectronic device for detecting an analyte within the body of an animal. In a general sense this device comprises a bioreporter that is operably positioned above a substrate that is on an integrated circuit. The bioreporter is capable of metabolizing the target analyte and emits light consequent to this metabolism when in contact with the analyte. The device further comprises a sensor closely positioned to the integrated circuit that detects the emitted light and generates an electrical signal in proportion to the amount of light generated by the bioreporter. Preferably the entire implantable device is contained within a biocompatible container that is implanted within the body of the animal in which the analyte detection is desired.
The biocompatible container may be comprised of silicon nitride, silicon oxide, or a suitable polymeric matrix, with exemplary matrices such as polyvinyl alcohol, poly-L-lysine, and alginate being particularly preferred. The polymeric matrix may also further comprise a microporous, mesh-reinforced or a filter-supported hydrogel.
In certain embodiments, it may also be desirable to provide a transparent, biocompatible, bioresistant separator that is operably positioned between the phototransducer and the bioreporter.
The bioreporter preferably comprises a plurality of eukaryotic or prokaryotic cells that produce a bioluminescent reporter polypeptide in response to the presence of the target analyte. Prokaryotic cells such as one or more strains of bacteria, and eukaryotic cells such as mammalian cells are particularly preferred. Exemplary mammalian cells are human cells such as islet xcex2-cells, immortal stem cells, or hepatic cells, with immortal stem cells being particularly preferred.
These cells preferably comprise one or more nucleic acid segments that encode a luciferase polypeptide or a green fluorescent protein that is produced by the cells in response to the presence of the analyte. Preferably the nucleic acid segment encodes an Aqueorea Victoria, Renilla reniformis, or a humanized green fluorescent protein, or more preferably, a bacterial Lux polypeptide, such as the LuxA, LuxB, LuxC, LuxD, or LuxE polypeptide, or the LuxAB or LuxCDE fused polypeptides described herein.
Exemplary bacterial lux gene sequences that may be employed to prepare the genetic constructs include the Vibrio fischerii or more preferably, the Xenorhabdus luminescens luxA, luxB, luxC, luxD, luxE, luxAB, or luxCDE genes.
Exemplary lux gene sequences that may be employed for preparation of the genetic constructs as described herein include the gene sequences disclosed in SEQ ID NO:1. Exemplary Lux polypeptide sequences are disclosed in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
The Lux polypeptides preferably comprise at least a 10 contiguous amino acid sequence from one or more of the polypeptide sequences disclosed in SEQ ID NO:2 through SEQ ID NO:6. More preferably the Lux polypeptides comprise at least a 15 contiguous amino acid sequence from one or more of the polypeptide sequences disclosed in SEQ ID NO:2 through SEQ ID NO:6, and more preferably still, comprise at least a 20 contiguous amino acid sequence from one or more of the polypeptide sequences disclosed in SEQ ID NO:2 through SEQ ID NO:6.
Such polypeptides are preferably encoded by a nucleic acid sequence that comprises at least 20, at least 25, at least 30, at least 35, at least 40, or at least 45 or more contiguous nucleotides from SEQ ID NO:1.
The expression of the Lux-encoding nucleic acid segments is preferably regulated by a nucleic acid regulatory sequence operably linked to the Lux-encoding segment. Preferably this regulatory sequence comprises a cis-acting element that is responsive to the presence of the target analyte. Exemplary cis-acting response elements are selected from the group consisting of an S14 gene sequence, a hepatic L-pyruvate kinase gene sequence, a hepatic 6-phosphofructo-2-kinase gene sequence, a xcex2-islets insulin gene sequence, a mesangial transforming growth factor-xcex2 gene sequence, and an acetyl-coenzyme-A carboxylase gene sequence.
In an illustrative embodiment, the cis-acting response element comprises a contiguous nucleotide sequence from a xcex2-islets insulin gene sequence or a hepatic L-pyruvate kinase gene sequence. Expression of the nucleic acid sequence is preferably regulated by a promoter sequence such as the one derived from an L-pyruvate kinase-encoding gene described herein.
The device may further comprise a wireless transmitter, an antenna, and a source of nutrients capable of sustaining the bioreporter cells. Likewise the biocompatible container enclosing the bioreporter may further comprise a membrane that is permeable to the analyte but not to the bioreporter cells themselves. Such a semi-permeable membrane permits analytes to flow freely from the bodily fluid into the detector device, but restricts the migration of bioreporter cells from the device into the surrounding tissues or circulatory system of the body in which the device is implanted.
In one embodiment, the integrated circuit is a complementary metal oxide semiconductor (CMOS) integrated circuit. The integrated circuit may comprise one or more phototransducer, that themselves may be comprised of one or more photodiodes. Likewise, the integrated circuit may also further comprise a photodiode, a current-to-frequency converter, a digital counter, and/or a transmitter that is capable of transmitting either digital or analog data.
The invention also provides an implantable controlled drug delivery system that comprises both the bioluminescent bioreporter integrated circuit (BBIC) device and an implantable drug delivery pump that is capable of being operably controlled by the BBIC and that is capable of delivering the drug to the body of the animal in response to controls by the device. The invention also concerns a method of providing a controlled supply of a drug to a patient in need thereof. The method generally involves implanting within the body of the patient the controlled drug delivery system.
The invention also provides a method of determining the amount of a drug required by a patient in need thereof, such as in the case of giving a diabetic patient an appropriate amount of insulin. The method generally involves implanting within the body of the diabetic patient one or more BBIC devices that are responsive to either glucose, glucagons, insulin, or another glucose metabolite, and determining the amount of insulin required by the patient based upon the levels of the analyte detected in the body fluids by. the device. When the device indicates that higher levels of insulin are required, the appropriate control signal can be sent to the drug delivery system and more insulin is injected into the body. When the device indicates that lower levels of insulin are required, then the appropriate control signal can be sent to the drug delivery system and less insulin can be administered. Such xe2x80x9creal-timexe2x80x9d monitoring of glucose in the body of the animal permits for controlled release of insulin throughout the day, and obviates the need for daily or more frequent injections of insulin that may either be too much or too little for the particular time of administration. This affords a more cost-effective administration of the drug, and also provides a more stable dosing of the insulin to the patient on an xe2x80x9cas neededxe2x80x9d basis.
The invention also provides a kit for the detection of an analyte, and such kits generally will include one or more of the disclosed BBIC devices in combination with appropriate instructions for using the detection device. Such kits may also routinely contain one or more standardized reference solutions for calibrating the device, and may also include suitable storage or nutrient medium for sustaining the bioreporter cells either during storage or during use once implanted within the body of the animal. In the case of therapeutic kits, such kits will also generally include one or more controlled delivery systems for administration of the drug to the body of the animal.
The invention also provides a method of regulating the blood glucose level of an animal in need thereof. This method generally comprises monitoring the level of glucose in the bloodstream or interstitial fluid of the patient using the BBIC device, and administering to the patient an effective amount of an insulin composition sufficient to regulate the blood glucose level.
This new type of bioluminescence-based bioreporter is capable of monitoring target substances without the disadvantageous requirement that cells be destroyed to produce the measurable signal. This allows for monitoring to occur continuously, on-line and in real-time (Simpson et al., 1998a, 1998b). These cells rely on luciferase genes (designated lux in prokaryotes and luc in eukaryotes) for the reporter enzyme system. U.S. patent application Ser. No. 08/978,439 and Intl. Pat. Appl. Ser. No. PCT/US98/25295 (each of which is specifically incorporated herein by reference in its entirety) disclose a self-contained miniature bioluminescence bioreporter integrated circuit (xe2x80x9cBBICxe2x80x9d) that was designed to detect specific molecular targets ex situ or ex vivo.
The present invention concerns an implantable, or an in situ or an in vivo BBIC device that is capable of being implanted within the body of an animal, and that is capable of detecting the concentration of one or more analytes present within the animal. The implantable monolithic bioelectronic device of the present invention generally comprises a substrate, a bioreporter capable of responding to a particular substance by the emission of light, a container affixed to the substrate capable of holding the bioreporter, an integrated circuit on the substrate including a phototransducer operative to generate an electrical signal in response to the light wherein the signal indicates the concentration of the substance; and a biocompatible housing that is capable of being implanted within the body of an animal, with that portion of the housing covering the bioreporter container comprising a semi-permeable membrane that permits passage of the analyte from the body of the animal to contact the biosensor, but restricts the bioreporter molecules from diffusing into the body of the animal that contains the implanted device. The bioreporter may be in solution, that is a cell suspension, and entrapped in the container by the semi-permeable membrane, or alternatively the bioreporter may be encapsulated in a selectively permeable polymer matrix that is capable of allowing the selected substance in solution reach the bioreporter. Preferably, the matrix is optically clear.
The apparatus may further comprise a layer of bioresistant/biocompatible material between the substrate and the container, such a layer of silicon nitride. The integrated Circuit is preferably a CMOS integrated circuit, and the phototransducer is preferably a photodiode.
The integrated circuit may also include a current to frequency converter and/or a digital counter. Additionally, the integrated circuit may also include one or more transmitters. Such transmitters may be wireless, or conventionally wired. In preferred embodiment, the apparatus also includes a drug delivery device capable of receiving transmissions from the transmitter.
A further embodiment of the invention is an implantable apparatus for detecting a selected substance in solution, which comprises an integrated circuit including a phototransducer adapted to input an electrical signal into the circuit in response to light, a bioreporter capable of responding to selected substance in solution by emitting light, the reporter adapted to contact the substance; and a transparent, biocompatible, and bioresistant separator positioned between the phototransducer and the bioreporter to enable light emitted from the bioreporter to strike the phototransducer. In a preferred embodiment of the present invention, the selected substance is glucose. The bioreporter may be a mammalian cell that contains a nucleotide sequence that encodes one or more luminescent reporter molecules. Such a nucelotide sequence may comprise one or more lux genes. In a preferred embodiment the lux genes comprise both luxCDE genes and fused luxAB genes. In one embodiment, these lux genes are derived from Xenorhabdus luminescens. The lux genes may be regulated by a nucleic acid sequence comprising one or more cis-acting glucose response elements. In an illustrative embodiment, the glucose response element may be derived from the xcex2-islets or hepatic L-pyruvate kinase gene. In a highly preferred embodiment the p.LPK.LucFF plasmid is used to provide one or more glucose response elements and the L-pyruvate kinase promoter to drive the expression of one or more lux genes. The cells constituting the bioreporter may be in suspension, entrapped in place on the IC by a semi-permeable membrane. Alternatively the cells constituting the bioreporter may be encapsulated in a polymer matrix affixed to IC. Such a matrix may be permeable to the selected substance in solution.
A further embodiment of the invention concerns an implantable monolithic bioelectronic device for detecting a selected substance in body fluid. This device generally comprises a biocompatible housing; a bioreporter capable of responding to a selected substance by emitting; and, a sensor capable of generating an electrical signal in response to the reception of the emitted light. Such a device may also include a transparent, bioresistant and biocompatible separator positioned between the bioreporter and the sensor and a semi-permeable membrane positioned in the biocompatible housing so that the selected substance can access the bioreporter.
A standard integrated circuit (IC) is coated with a layer of insulating material such as silicon dioxide or silicon nitride. This process is called passivation and serves to protect the surface of the chip from moisture, contamination, and mechanical damage. BBICs require a second coating that must be biocompatible and bioresistant, must protect the OASIC from chemical stresses, must be optically tuned to efficiently transmit the light from the material under test, must adhere to an oxide coating, must be pin-hole free, and must be able to be patterned in order to form openings over the bonding pads and whatever structures that might be needed to maintain the bioreporter or collect a sample.
The present invention contemplates that the components of the biosensor may be packaged in kit form. Kits may comprise, in suitable container means, one or more bioreporters and an integrated circuit including a phototransducer. Kits may further comprise a drug delivery device.